Nucleic acid sequencing by raman monitoring of uptake of precursors during molecular replication

ABSTRACT

The methods, compositions and apparatus disclosed herein are of use for nucleic acid sequence determination. The methods involve isolation of one or more nucleic acid template molecules and polymerization of a nascent complementary strand of nucleic acid, using a DNA or RNA polymerase or similar synthetic reagent. As the nascent strand is extended one nucleotide at a time, the disappearance of nucleotide precursors from solution is monitored by Raman spectroscopy or FRET. The nucleic acid sequence of the nascent strand, and the complementary sequence of the template strand, may be determined by tracking the order of incorporation of nucleotide precursors during the polymerization reaction. Certain embodiments concern apparatus comprising a reaction chamber and detection unit, of use in practicing the claimed methods. The methods, compositions and apparatus are of use in sequencing very long nucleic acid templates in a single sequencing reaction.

FIELD OF THE INVENTION

The present methods, compositions and apparatus relate to the fields ofmolecular biology and genomics. More particularly, the disclosedmethods, compositions and apparatus concern nucleic acid sequencing.

BACKGROUND

The advent of the human genome project required that improved methodsfor sequencing nucleic acids, such as DNA (deoxyribonucleic acid) andRNA (ribonucleic acid), be developed. Genetic information is stored inthe form of very long molecules of DNA organized into chromosomes. Thetwenty-three pairs of chromosomes in the human genome containapproximately three billion bases of DNA sequence. This DNA sequenceinformation determines multiple characteristics of each individual, suchas height, eye color and ethnicity. Many common diseases, such ascancer, cystic fibrosis, sickle cell anemia and muscular dystrophy arebased at least in part on variations in DNA sequence.

Determination of the entire sequence of the human genome has provided afoundation for identifying the genetic basis of such diseases. However,a great deal of work remains to be done to identify the geneticvariations associated with each disease. That would require DNAsequencing of portions of chromosomes in individuals or familiesexhibiting each such disease, in order to identify specific changes inDNA sequence that promote the disease. RNA, an intermediary moleculerequired for processing of genetic information, can also be sequenced insome cases to identify the genetic bases of various diseases.

Existing methods for nucleic acid sequencing, based on detection offluorescently labeled nucleic acids that have been separated by size,are limited by the length of the nucleic acid that can be sequenced.Typically, only 500 to 1,000 bases of nucleic acid sequence can bedetermined at one time. This is much shorter than the length of thefunctional unit of DNA, referred to as a gene, which can be tens or evenhundreds of thousands of bases in length. Using current methods,determination of a complete gene sequence requires that many copies ofthe gene be produced, cut into overlapping fragments and sequenced,after which the overlapping DNA sequences may be assembled into thecomplete gene. This process is laborious, expensive, inefficient andtime-consuming.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain embodiments. Those embodimentsmay be better understood by reference to one or more of these drawingsin combination with the detailed description of specific embodimentspresented herein.

FIG. 1 illustrates an exemplary apparatus 10 (not to scale) and methodfor DNA sequencing in which a nucleic acid 13 is sequenced by monitoringthe uptake of nucleotide precursors 17 from solution during nucleic acidsynthesis.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The disclosed methods, compositions and apparatus are of use for therapid, automated sequencing of nucleic acids 13. In particularembodiments, the methods, compositions and apparatus are suitable forobtaining the sequences of very long nucleic acid 13 molecules ofgreater than 1,000, greater than 2,000, greater than 5,000, greater than10,000 greater than 20,000, greater than 50,000, greater than 100,000 oreven more bases in length. In various embodiments, such sequenceinformation may be obtained during the course of a single sequencingrun, using one molecule of template nucleic acid 13. In otherembodiments, multiple copies of the template nucleic acid molecule 13may be sequenced in parallel or sequentially to confirm the nucleic acid13 sequence or to obtain complete sequence data. In alternativeembodiments, both the template strand 13 and its complementary strandmay be sequenced to confirm the accuracy of the sequence information.Advantages over prior methods of nucleic acid 13 sequencing include theability to read long nucleic acid 13 sequences in a single sequencingrun, greater speed of obtaining sequence data, decreased cost ofsequencing and greater efficiency in terms of the amount of operatortime required per unit of sequence data generated.

In certain embodiments, the nucleic acid 13 to be sequenced is DNA,although it is contemplated that other nucleic acids 13 comprising RNAor synthetic nucleotide analogs could be sequenced as well. Thefollowing detailed description contains numerous specific details inorder to provide a more thorough understanding of the disclosedembodiments. However, it will be apparent to those skilled in the artthat the embodiments may be practiced without these specific details. Inother instances, those devices, methods, procedures, and individualcomponents that are well known in the art have not been described indetail herein.

Certain embodiments are illustrated in FIG. 1. FIG. 1 shows an apparatus10 for nucleic acid 13 sequencing comprising a reaction chamber 11 and adetection unit 12. The reaction chamber 11 contains a nucleic acid(template) molecule 13 attached to an immobilization surface 14 alongwith a synthetic reagent 15, such as a DNA polymerase. A primer molecule16 that is complementary in sequence to the template molecule 13 isallowed to hybridize to the template molecule 13. Nucleotide precursors17 are present in solution in the reaction chamber 11. For synthesis ofa nascent DNA strand 16, the nucleotide precursors 17 must include atleast one molecule each of deoxyadenosine-5′-triphosphate (dATP),deoxyguanosine-5′-triphosphate (dGTP), deoxycytosine-5′-triphosphate(dCTP) and deoxythymidine-5′-triphosphate (dTTP). For synthesis of anascent RNA strand 16, the nucleotide precursors 17 must comprise ATP,CTP, GTP and uridine-5′-triphosphate (UTP).

To initiate a sequencing reaction, the polymerase 15 adds one nucleotideprecursor molecule 17 at a time to the 3′ end of the primer 16,elongating the primer molecule 16. As the primer molecule 16 isextended, it is referred to as a nascent strand 16. For each round ofelongation, a single nucleotide precursor 17 is incorporated into thenascent strand 16. Because incorporation of nucleotide precursors 17 isdetermined by Watson-Crick base pair interactions with the templatestrand 13, the sequence of the growing nascent strand 16 will becomplementary to the sequence of the template strand 13. In Watson-Crickbase pairing, an adenosine (A) residue on one strand is always pairedwith a thymidine (T) residue on the other strand, or a uridine (U)residue if the strand is RNA. Similarly, a guanosine (G) residue on onestrand is always paired with a cytosine (C) residue on the other strand.Thus, the sequence of the template strand 13 may be determined from thesequence of the nascent strand 16.

FIG. 1 illustrates embodiments in which a single nucleic acid molecule13 is contained in a single reaction chamber 11. In alternativeembodiments, multiple nucleic acid molecules 13, each in a separatereaction chamber 11, may be sequenced simultaneously. In such cases, thenucleic acid template 13 in each reaction chamber 11 may be identical ormay be different. In other alternative embodiments, two or more templatenucleic acid molecules 13 may be present in a single reaction chamber11. In such embodiments, the nucleic acid molecules 13 will be identicalin sequence. Where more than one template nucleic acid 13 is present inthe reaction chamber 11, the Raman emission signals will represent anaverage of the nucleic acid precursors 17 incorporated into all nascentstrands 16 in the reaction chamber 11. The skilled artisan will be ableto correct the signal obtained at any given time for synthetic reactionsthat either lag behind or precede the majority of reactions occurring inthe reaction chamber 11, using known data analysis techniques.

The skilled artisan will realize that depending on the polymerasemolecule 15 used, the nascent strand 16 may contain some percentage ofmismatched bases, where the newly incorporated base is not correctlyhydrogen bonded with the corresponding base in the template strand 13.In various embodiments, an accuracy of at least 90%, at least 95%, atleast 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9%or higher may be observed. The skilled artisan will be aware thatcertain polymerases 15 have an error correction activity (also referredto as a 3′ exonuclease or proof-reading activity) that acts to remove anewly incorporated nucleotide precursor 17 that is incorrectlybase-paired to the template strand 13. In various embodiments,polymerases 15 with or without a proof-reading activity may be employed.The skilled artisan will also be aware that certain polymerases 15, suchas reverse transcriptase, have an inherently high error rate, allowingfrequent incorporation of mismatched bases. Depending on the embodiment,a polymerase 15 with either a higher or a lower inherent error rate maybe selected. In certain embodiments, a polymerase 15 with the lowestpossible error rate may be used. Polymerase 15 error rates are known inthe art.

The detection unit 12 comprises an excitation source 18, such as alaser, and a Raman spectroscopy detector 19. The excitation source 18illuminates the reaction chamber 11 with an excitation beam 20. Theexcitation beam 20 interacts with the nucleotide precursors 17,resulting in the excitation of electrons to a higher energy state. Asthe electrons return to a lower energy state, they emit a Raman emissionsignal that is detected by the Raman detector 19. Because the Ramanemission signal from each of the four types of nucleotide precursor 17can be distinguished, the detection unit 12 is capable of measuring theamount of each type of nucleotide precursor 17 in the reaction chamber11.

The incorporation of nucleotide precursors 17 into the growing nascentstrand 16 results in a depletion of nucleotide precursors 17 from thereaction chamber 11. In order for the synthetic reaction to continue, asource of fresh nucleotide precursors 17 may be required. This source isshown in FIG. 1 as a molecule dispenser 21. In alternative embodiments,a molecule dispenser 21 may or may not be part of the sequencingapparatus 10.

In certain embodiments, the molecule dispenser 21 is designed to releaseeach of the four nucleotide precursors 17 in equal amounts, calibratedto the rate of synthesis of the nascent strand 16. However, nucleicacids 13 do not necessarily exhibit a uniform distribution of A, T, Gand C residues. In particular, certain regions of DNA molecules may beeither AT rich or GC rich, depending on the species from which the DNAis obtained and the specific region of the DNA molecule being sequenced.In alternative embodiments, the release of nucleotide precursors 17 fromthe molecule dispenser 21 is controlled, so that relatively constantconcentrations of each type of nucleotide precursor 17 are maintained inthe reaction chamber 11. Such embodiments may utilize an informationprocessing and control system that interfaces between the detection unit12 and the molecule dispenser 21.

In embodiments involving an information processing and control system,such as a computer or microprocessor attached to or incorporating a datastorage unit, data may be collected from a detector 19, such as aspectrometer or a monochromator array. The information processing andcontrol system may maintain a database associating specific Ramansignatures with specific nucleotide precursors 17. The informationprocessing and control system may record the signatures detected by thedetector 19 and may correlate those signatures with the signatures ofknown nucleotide precursors 17. The information processing and controlsystem may also maintain a record of nucleotide precursor 17 uptake thatindicates the sequence of the template molecule 13. The informationprocessing and control system may also perform standard procedures knownin the art, such as subtraction of background signals.

In embodiments involving a molecule dispenser 21, the addition ofnucleotide precursors 17 to the reaction chamber 11, simultaneously withthe incorporation of nucleotide precursors 17 into the nascent strand 16may result in a complex Raman signal. In particular embodiments, thesynthetic reaction may be allowed to run to completion or close tocompletion before additional nucleotide precursors 17 are added to thereaction chamber 11. In alternative embodiments, the addition ofnucleotide precursors 17 to the reaction chamber 11 may occursimultaneously with incorporation of nucleotide precursors 17 into thenascent strand 16. In such embodiments, the information processing andcontrol system may be used to correct the data on nucleotide precursor17 concentration obtained from the Raman emission spectrum for theamount of nucleotide precursors 17 added by the molecule dispenser 21.

In certain embodiments, the reaction chamber 11 may contain a singlemolecule of each type of nucleotide precursor 17. In such embodiments,the release of nucleotide precursors 17 from the molecule dispenser 21may be tightly linked to the incorporation of nucleotide precursors 17into the nascent strand 16, in order to avoid delays in the syntheticreaction due to the absence of a required nucleotide precursor 17.

Certain embodiments concern synthesis of a nascent strand 16 of DNA. Thetemplate strand 13 can be either RNA or DNA. With an RNA template strand13, the synthetic reagent 15 may be a reverse transcriptase, examples ofwhich are known in the art. In embodiments where the template strand 13is a molecule of DNA, the synthetic reagent 15 may be a DNA polymerase,examples of which are known in the art.

In other embodiments, the nascent strand 16 can be a molecule of RNA.This requires that the synthetic reagent 15 be an RNA polymerase. Inthese embodiments, no primer 16 is required. However, the templatestrand 13 must contain a promoter sequence that is effective to bind RNApolymerase 15 and initiate transcription of an RNA nascent strand 16.The exact composition of the promoter sequence depends on the type ofRNA polymerase 15 used. Optimization of promoter sequences to allow forefficient initiation of transcription is within the skill in the art.The embodiments are not limited as to the type of template molecule 13used, the type of nascent strand 16 synthesized, or the type ofpolymerase 15 utilized. Virtually any template 13 and any polymerase 15that can support synthesis of a nucleic acid molecule 16 complementaryin sequence to the template strand 13 may be used.

In some alternative embodiments, the nucleotide precursors 17 may bechemically modified with a tag. The tag has a unique and highly visibleoptical signature that can be distinguished for each of the commonnucleotide precursors 17. In certain embodiments, the tag may serve toincrease the strength of the Raman emission signal or to otherwiseenhance the sensitivity or specificity of the Raman detector 19 fornucleotide precursors 17. Non-limiting examples of tag molecules thatcould be used for embodiments involving Raman spectroscopy include TRIT(tetramethyl rhodamine isothiol), NBD (7-nitrobenz-2-oxa-1,3-diazole),Texas Red dye, phthalic acid, terephthalic acid, isophthalic acid,cresyl fast violet, cresyl blue violet, brilliant cresyl blue,para-aminobenzoic acid, erythrosine and aminoacridine. Other tagmoieties that may be of use for particular embodiments include cyanide,thiol, chlorine, bromine, methyl, phosphorus and sulfur. In certainembodiments, carbon nanotubes may be of use as Raman tags. The use oftags in Raman spectroscopy is known in the art (e.g., U.S. Pat. Nos.5,306,403 and 6,174,677). The skilled artisan will realize that Ramantags should generate distinguishable Raman spectra when bound todifferent nucleotide precursors 17, or different labels should bedesigned to bind only one type of nucleotide precursor 17.

In some embodiments, the tag exhibits an enhanced Raman signal. Inalternative embodiments, tags that exhibit other types of signals, suchas fluorescent or luminescent signals, may be employed. It iscontemplated that alternative methods of detection may be used in suchembodiments, for example fluorescence spectroscopy or luminescencespectroscopy. Many alternative methods of detection of nucleotideprecursors 17 in solution are known in the art and may be used. For suchmethods, the Raman spectroscopic detector 19 may be replaced with adetector 19 designed to detect fluorescence, luminescence or other typesof signals known in the art.

In certain embodiments, the template molecule 13 may be attached to asurface 14 such as functionalized glass, silicon, PDMS (polydimethlylsiloxane), silver or other metal coated surfaces, quartz, plastic, PTFE(polytetrafluoroethylene), PVP (polyvinyl pyrrolidone), polystyrene,polypropylene, polyacrylamide, latex, nylon, nitrocellulose, a glassbead, a magnetic bead, or any other material known in the art that iscapable of having functional groups such as amino, carboxyl, thiol,hydroxyl or Diels-Alder reactants incorporated on its surface.

In some embodiments, functional groups may be covalently attached tocross-linking agents so that binding interactions between templatestrand 13 and polymerase 15 may occur without steric hindrance. Typicalcross-linking groups include ethylene glycol oligomers and diamines.Attachment may be by either covalent or non-covalent binding. Variousmethods of attaching nucleic acid molecules 13 to surfaces 14 are knownin the art and may be employed.

Definitions

As used herein, “a” or “an” may mean one or more than one of an item.

“Nucleic acid” 13 means either DNA, RNA, single-stranded,double-stranded or triple stranded and any chemical modificationsthereof, although single-stranded nucleic acids 13 are preferred.Virtually any modification of the nucleic acid 13 is contemplated. Asused herein, a single stranded nucleic acid 13 may be denoted by theprefix “ss”, a double stranded nucleic acid by the prefix “ds”, and atriple stranded nucleic acid by the prefix “ts.”

A “nucleic acid” 13 may be of almost any length, from 10, 20, 30, 40,50, 60, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, 500, 600,700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000,6000, 7000, 8000, 9000, 10,000, 15,000, 20,000, 30,000, 40,000, 50,000,75,000, 100,000, 150,000, 200,000, 500,000, 1,000,000, 1,500,000,2,000,000, 5,000,000 or even more bases in length, up to a full-lengthchromosomal DNA molecule 13.

A “nucleoside” is a molecule comprising a base (A, T, G, C or U)covalently attached to a pentose sugar such as deoxyribose, ribose orderivatives or analogs of pentose sugars.

A “nucleotide” refers to a nucleoside further comprising at least onephosphate group covalently attached to the pentose sugar. In someembodiments, the nucleotide precursors 17 are ribonucleosidetriphosphates or deoxyribonucleoside triphosphates. It is contemplatedthat various substitutions or modifications may be made in the structureof the nucleotide precursors 17, so long as they are still capable ofbeing incorporated into the nascent strand 16 by the polymerase 15. Forexample, in certain embodiments the ribose or deoxyribose moiety may besubstituted with another pentose sugar or a pentose sugar analog. Inother embodiments, the phosphate groups may be substituted by variousgroups, such as phosphonates, sulphates or sulfonates. In still otherembodiments, the purine or pyrimidine bases may be substituted by otherpurines or pyrimidines or analogs thereof, so long as the sequence ofnucleotide precursors 17 incorporated into the nascent strand 16reflects the sequence of the template strand 13.

Nucleic Acids

Template molecules 13 may be prepared by any technique known to one ofordinary skill in the art. In certain embodiments, the templatemolecules 13 are naturally occurring DNA or RNA molecules, for example,chromosomal DNA or messenger RNA (mRNA). Virtually any naturallyoccurring nucleic acid 13 may be prepared and sequenced by the disclosedmethods including, without limit, chromosomal, mitochondrial orchloroplast DNA or ribosomal, transfer, heterogeneous nuclear ormessenger RNA. Nucleic acids 13 to be sequenced may be obtained fromeither prokaryotic or eukaryotic sources by standard methods known inthe art.

Methods for preparing and isolating various forms of cellular nucleicacids 13 are known. (See, e.g., Guide to Molecular Cloning Techniques,eds. Berger and Kimmel, Academic Press, New York, N.Y., 1987; MolecularCloning: A Laboratory Manual, 2nd Ed., eds. Sambrook, Fritsch andManiatis, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989).Generally, cells, tissues or other source material containing nucleicacids 13 to be sequenced are first homogenized, for example by freezingin liquid nitrogen followed by grinding in a morter and pestle. Certaintissues may be homogenized using a Waring blender, Virtis homogenizer,Dounce homogenizer or other homogenizer. Crude homogenates may beextracted with detergents, such as sodium dodecyl sulphate (SDS), TritonX-100, CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate), octylglucoside or other detergents known in the art.Alternatively or in addition, extraction may use chaotrophic agents suchas guanidinium isothiocyanate, or organic solvents such as phenol. Insome embodiments, protease treatment, for example with proteinase K, maybe used to degrade cell proteins. Particulate contaminants may beremoved by centrifugation or ultracentrifugation (for example, 10 to 30min at about 5,000 to 10,000×g, or 30 to 60 min at about 50,000 to100,000×g). Dialysis against aqueous buffer of low ionic strength may beof use to remove salts or other soluble contaminants. Nucleic acids 13may be precipitated by addition of ethanol at −20° C., or by addition ofsodium acetate (pH 6.5, about 0.3 M) and 0.8 volumes of 2-propanol.Precipitated nucleic acids 13 may be collected by centrifugation or, forchromosomal DNA, by spooling the precipitated DNA on a glass pipet orother probe.

The skilled artisan will realize that the procedures listed above areexemplary only and that many variations may be used, depending on theparticular type of nucleic acid 13 to be sequenced. For example,mitochondrial DNA is often prepared by cesium chloride density gradientcentrifugation, using step gradients, while mRNA is often prepared usingpreparative columns from commercial sources, such as Promega (Madison,Wis.) or Clontech (Palo Alto, Calif.). Such variations are known in theart.

The skilled artisan will realize that depending on the type of templatenucleic acid 13 to be prepared, various nuclease inhibitors may be used.For example, RNase contamination in bulk solutions may be eliminated bytreatment with diethyl pyrocarbonate (DEPC), while commerciallyavailable nuclease inhibitors may be obtained from standard sources suchas Promega (Madison, Wis.) or BRL (Gaithersburg, Md.). Purified nucleicacid 13 may be dissolved in aqueous buffer, such as TE (Tris-EDTA)(ethylene diamine tetraacetic acid) and stored at −20° C. or in liquidnitrogen prior to use.

In cases where single stranded DNA (ssDNA) 13 is to be sequenced, assDNA 13 may be prepared from double stranded DNA (dsDNA) by standardmethods. Most simply, dsDNA may be heated above its annealingtemperature, at which point it spontaneously separates into ssDNA 13.Representative conditions might involve heating at 92 to 95° C. for 5min or longer. Formulas for determining conditions to separate dsDNA,based for example on GC content and the length of the molecule, areknown in the art. Alternatively, single-stranded DNA 13 may be preparedfrom double-stranded DNA by standard amplification techniques known inthe art, using a primer that only binds to one strand of double-strandedDNA. Other methods of preparing single-stranded DNA 13 are known in theart, for example by inserting the double-stranded nucleic acid to besequenced into the replicative form of a phage like M13, and allowingthe phage to produce single-stranded copies of the template 13.

Although certain embodiments concern preparation of naturally occurringnucleic acids 13, virtually any type of nucleic acid 13 that can serveas a template for an RNA or DNA polymerase 15 could potentially besequenced. For example, nucleic acids 13 prepared by variousamplification techniques, such as polymerase chain reaction (PCR™)amplification, could be sequenced. (See U.S. Pat. Nos. 4,683,195,4,683,202 and 4,800,159.) Nucleic acids 13 to be sequenced mayalternatively be cloned in standard vectors, such as plasmids, cosmids,BACs (bacterial artificial chromosomes) or YACs (yeast artificialchromosomes). (See, e.g., Berger and Kimmel, 1987; Sambrook et al.,1989.) Nucleic acid inserts 13 may be isolated from vector DNA, forexample, by excision with appropriate restriction endonucleases,followed by agarose gel electrophoresis and ethidium bromide staining.Selected size-fractionated nucleic acids 13 may be removed from gels,for example by the use of low melting point agarose or by electroelutionfrom gel slices. Methods for insert isolation are known to the person ofordinary skill in the art.

Isolation of Single Nucleic Acid Molecules

In certain embodiments, the nucleic acid molecule 13 to be sequenced isa single molecule of ssDNA or ssRNA. A variety of methods for selectionand manipulation of single ssDNA or ssRNA molecules 13 may be used, forexample, hydrodynamic focusing, micro-manipulator coupling, opticaltrapping, or combination of these and similar methods. (See, e.g.,Goodwin et al., 1996, Acc. Chem. Res. 29:607-619; U.S. Pat. Nos.4,962,037; 5,405,747; 5,776,674; 6,136,543; 6,225,068.)

In certain embodiments, microfluidics or nanofluidics may be used tosort and isolate template nucleic acids 13. Hydrodynamics may be used tomanipulate the movement of nucleic acids 13 into a microchannel,microcapillary, or a micropore. In one embodiment, hydrodynamic forcesmay be used to move nucleic acid molecules 13 across a comb structure toseparate single nucleic acid molecules 13. Once the nucleic acidmolecules 13 have been separated, hydrodynamic focusing may be used toposition the molecules 13. A thermal or electric potential, pressure orvacuum can also be used to provide a motive force for manipulation ofnucleic acids 13. In exemplary embodiments, manipulation of templatenucleic acids 13 for sequencing may involve the use of a channel blockdesign incorporating microfabricated channels and an integrated gelmaterial, as disclosed in U.S. Pat. Nos. 5,867,266 and 6,214,246.

In another embodiment, a sample containing the nucleic acid template 13may be diluted prior to coupling to an immobilization surface 14. Inexemplary embodiments, the immobilization surface 14 may be in the formof magnetic or non-magnetic beads or other discrete structural units. Atan appropriate dilution, each bead will have a statistical probabilityof binding zero or one nucleic acid molecules 13. Beads with oneattached nucleic acid molecule 13 may be identified using, for example,fluorescent dyes and flow cytometer sorting or magnetic sorting.Depending on the relative sizes and uniformity of the beads and thenucleic acids 13, it may be possible to use a magnetic filter and massseparation to separate beads containing a single bound nucleic acidmolecule 13. In other embodiments, multiple nucleic acids 13 attached toa single bead or other immobilization surface 14 may be sequenced.

In alternative embodiments, a coated fiber tip 14 may be used togenerate single molecule nucleic acid templates 13 for sequencing (e.g.,U.S. Pat. No. 6,225,068). In other alternative embodiments, theimmobilization surfaces 14 may be prepared to contain a single moleculeof avidin or other cross-linking agent. Such a surface 14 could attach asingle biotinylated primer 16, which in turn can hybridize with a singletemplate nucleic acid 13 to be sequenced. This embodiment is not limitedto the avidin-biotin binding system, but may be adapted to any couplingsystem known in the art.

In other alternative embodiments, an optical trap may be used formanipulation of single molecule nucleic acid templates 13 forsequencing. (E.g., U.S. Pat. No. 5,776,674). Exemplary optical trappingsystems are commercially available from Cell Robotics, Inc.(Albuquerque, N. Mex), S+L GmbH (Heidelberg, Germany) and P.A.L.M. Gmbh(Wolfratshausen, Germany).

Methods of Immobilization

In various embodiments, the nucleic acid molecules 13 to be sequencedmay be attached to a solid surface 14 (or immobilized). Immobilizationof nucleic acid molecules 13 may be achieved by a variety of methodsinvolving either non-covalent or covalent attachment between the nucleicacid molecule 13 and the surface 14. In an exemplary embodiment,immobilization may be achieved by coating a surface 14 with streptavidinor avidin and the subsequent attachment of a biotinylated polynucleotide13 (Holmstrom et al., Anal. Biochem. 209:278-283, 1993). Immobilizationmay also occur by coating a silicon, glass or other surface 14 withpoly-L-Lys (lysine) or poly L-Lys, Phe (phenylalanine), followed bycovalent attachment of either amino- or sulfhydryl-modified nucleicacids 13 using bifunctional crosslinking reagents (Running et al.,BioTechniques 8:276-277, 1990; Newton et al., Nucleic Acids Res.21:1155-62, 1993). Amine residues may be introduced onto a surface 14through the use of aminosilane for cross-linking.

Immobilization may take place by direct covalent attachment of5′-phosphorylated nucleic acids 13 to chemically modified surfaces 14(Rasmussen et al., Anal. Biochem. 198:138-142, 1991). The covalent bondbetween the nucleic acid 13 and the surface 14 is formed by condensationwith a water-soluble carbodiimide. This method facilitates apredominantly 5′-attachment of the nucleic acids 13 via their5′-phosphates.

DNA 13 is commonly bound to glass by first silanizing the glass surface14, then activating with carbodiimide or glutaraldehyde. Alternativeprocedures may use reagents such as 3-glycidoxypropyltrimethoxysilane(GOP) or aminopropyltrimethoxysilane (APTS) with DNA 13 linked via aminolinkers incorporated either at the 3′ or 5′ end of the molecule. DNA 13may be bound directly to membrane surfaces 14 using ultravioletradiation. Other non-limiting examples of immobilization techniques fornucleic acids 13 are disclosed in U.S. Pat. Nos. 5,610,287, 5,776,674and 6,225,068.

The type of surface 14 to be used for immobilization of the nucleic acid13 is not limiting. In various embodiments, the immobilization surface14 may be magnetic beads, non-magnetic beads, a planar surface, apointed surface, or any other conformation of solid surface 14comprising almost any material, so long as the material is sufficientlydurable and inert to allow the nucleic acid 13 sequencing reaction tooccur. Non-limiting examples of surfaces 14 that may be used includeglass, silica, silicate, PDMS, silver or other metal coated surfaces,nitrocellulose, nylon, activated quartz, activated glass, polyvinylidenedifluoride (PVDF), polystyrene, polyacrylamide, other polymers such aspoly(vinyl chloride), poly(methyl methacrylate) or poly(dimethylsiloxane), and photopolymers which contain photoreactive species such asnitrenes, carbenes and ketyl radicals capable of forming covalent linkswith nucleic acid molecules 13 (See U.S. Pat. Nos. 5,405,766 and5,986,076).

Bifunctional cross-linking reagents may be of use in variousembodiments, such as attaching a nucleic acid molecule 13 to a surface14. The bifunctional cross-linking reagents can be divided according tothe specificity of their functional groups, e.g., amino, guanidino,indole, or carboxyl specific groups. Of these, reagents directed to freeamino groups are popular because of their commercial availability, easeof synthesis and the mild reaction conditions under which they can beapplied. Exemplary methods for cross-linking molecules are disclosed inU.S. Pat. Nos. 5,603,872 and 5,401,511. Cross-linking reagents includeglutaraldehyde (GAD), bifunctional oxirane (OXR), ethylene glycoldiglycidyl ether (EGDE), and carbodiimides, such as1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC).

Synthetic Reagent

In certain embodiments, the sequencing reaction involves binding of asynthetic reagent 15, such as a DNA polymerase 15, to a primer molecule16 and the catalyzed addition of nucleotide precursors 17 to the 3′ endof the primer 16. Non-limiting examples of synthetic reagents 15 ofpotential use include DNA polymerases, RNA polymerases, reversetranscriptases, and RNA-dependent RNA polymerases. The differencesbetween these synthetic reagents 15 in terms of their “proofreading”activity and requirement or lack of requirement for primers and promotersequences are discussed herein and are known in the art. Where RNApolymerases are used as the synthetic reagent 15, the template molecule13 to be sequenced may be double-stranded DNA.

In embodiments using synthetic reagents 15 with proofreading capability,the release of incorrectly incorporated nucleotide precursors 17 isdetected by the detection unit 12, and the sequence data is accordinglycorrected. In embodiments using synthetic reagents 15 withoutproofreading capability, errors are not corrected. These errors can beeliminated by sequencing both strands of the original template 13, or bysequencing multiple copies of the same strand 13. Non-limiting examplesof polymerases 15 that could be used include Thermatoga maritima DNApolymerase, AmplitaqFS™ DNA polymerase, Taquenase™ DNA polymerase,ThermoSequenase™, Taq DNA polymerase, Qbeta™ replicase, T4 DNApolymerase, Thermus thermophilus DNA polymerase, RNA-dependent RNApolymerase and SP6 RNA polymerase.

A number of synthetic reagents 15 are commercially available, includingPwo DNA Polymerase from Boehringer Mannheim Biochemicals (Indianapolis,Ind.); Bst Polymerase from Bio-Rad Laboratories (Hercules, Calif.);IsoTherm™ DNA Polymerase from Epicentre Technologies (Madison, Wis.);Moloney Murine Leukemia Virus Reverse Transcriptase, Pfu DNA Polymerase,Avian Myeloblastosis Virus Reverse Transcriptase, Thermus flavus (Tfl)DNA Polymerase and Thermococcus litoralis (Tli) DNA Polymerase fromPromega (Madison, Wis.); RAV2 Reverse Transcriptase, HIV-1 ReverseTranscriptase, T7 RNA Polymerase, T3 RNA Polymerase, SP6 RNA Polymerase,RNA Polymerase E. coli, Thermus aquaticus DNA Polymerase, T7 DNAPolymerase +/−3′→5′ exonuclease, Klenow Fragment of DNA Polymerase I,Thermus ‘ubiquitous’ DNA Polymerase, and DNA polymerase I from AmershamPharmacia Biotech (Piscataway, N.J.). However, any synthetic reagent 15that is known in the art for the template dependent polymerization ofnucleotide precursors 17 may be used. (See, e.g., Goodman and Tippin,Nat. Rev. Mol. Cell Biol. 1(2):101-9, 2000; U.S. Pat. No. 6,090,589.)

The skilled artisan will realize that the rate of polymerase 15 activitymay be manipulated to coincide with the optimal rate of analysis ofnucleotide precursors 17 by the detection unit 12. Various methods areknown for adjusting the rate of polymerase 15 activity, includingadjusting the temperature, pressure, pH, salt concentration, divalentcation concentration, or the concentration of nucleotide precursors 17in the reaction chamber 11. Methods of optimization of polymerase 15activity are known to the person of ordinary skill in the art.

Labels

Certain embodiments may involve incorporating a label into thenucleotide precursors 17, to facilitate their measurement by thedetection unit 12. A number of different labels may be used, such asRaman tags, fluorophores, chromophores, radioisotopes, enzymatic tags,antibodies, chemiluminescent, electroluminescent, affinity labels, etc.One of skill in the art will recognize that these and other labelmoieties not mentioned herein can be used in the disclosed methods.

Labels for use in embodiments involving Raman spectroscopy are discussedabove. In other embodiments, the label moiety to be used may be afluorophore, such as Alexa 350, Alexa 430, AMCA(7-amino-4-methylcoumarin-3-acetic acid), BODIPY(5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid) 630/650,BODIPY 650/665, BODIPY-FL (fluorescein), BODIPY-R6G(6-carboxyrhodamine), BODIPY-TMR (tetramethylrhodamine), BODIPY-TRX(Texas Red-X), Cascade Blue, Cy2 (cyanine), Cy3, Cy5,6-FAM(5-carboxyfluorescein), Fluorescein, 6-JOE(2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein), Oregon Green 488,Oregon Green 500, Oregon Green 514, Pacific Blue, Rhodamine Green,Rhodamine Red, ROX (6-carboxy-X-rhodamine), TAMRA(N,N,N′,N′-tetramethyl-6-carboxyrhodamine), Tetramethylrhodamine, andTexas Red. Fluorescent or luminescent labels can be obtained fromstandard commercial sources, such as Molecular Probes (Eugene, Oreg.).

Primers

Primers 16 may be obtained by any method known in the art. Generally,primers 16 are between ten and twenty bases in length, although longerprimers 16 may be employed. In certain embodiments, primers 16 aredesigned to be exactly complementary in sequence to a known portion of atemplate nucleic acid molecule 13, preferably close to the attachmentsite of the template 13 to the immobilization surface 14. Methods forsynthesis of primers 16 of any sequence, for example using an automatednucleic acid synthesizer employing phosphoramidite chemistry are knownand such instruments may be obtained from standard sources, such asApplied Biosystems (Foster City, Calif.) or Millipore Corp. (Bedford,Mass.).

Other embodiments, involve sequencing a nucleic acid 13 in the absenceof a known primer binding site. In such cases, it may be possible to userandom primers 16, such as random hexamers or random oligomers of 7, 8,9, 10, 11, 12, 13, 14, 15 bases or greater length, to initiatepolymerization of a nascent strand 16. To avoid having multiplepolymerization sites on a single template strand 13, primers 16 besidesthose hybridized to the template molecule 13 near its attachment site tothe immobilization surface 14 may be removed before initiating thesynthetic reaction.

This could be accomplished, for example, by using an immobilizationsurface 14 coated with a binding agent, such as streptavidin. Acomplementary binding agent, such as biotin, could be attached to the 5′end of the primer molecules 16. After allowing hybridization betweenprimer 16 and template 13 to occur, those primer molecules 16 that arenot also bound to the immobilization surface 14 could be removed. Onlythose primers 16 that are hybridized to the template strand 13 willserve as primers 16 for template dependent DNA synthesis. In otheralternative embodiments, multiple primer molecules 16 may be attached tothe immobilization surface 14. A template molecule 13 is added andallowed to hydrogen bond to a complementary primer 16. A templatedependent polymerase 15 then acts to initiate nascent strand 16synthesis.

Other types of cross-linking could be used to selectively retain onlyone primer 16 per template strand 13, such as photoactivatablecross-linkers. As discussed above, a number of cross-linking agents areknown in the art and may be used. Cross-linking agents may also beattached to the immobilization surface 14 through linker arms, to avoidthe possibility of steric hindrance with the immobilization surface 14interfering with hydrogen bonding between the primer 16 and template 13.

Reaction Chamber

The reaction chamber 11 is designed to hold the immobilization surface14, nucleic acid template 13, primer 16, synthetic reagent 15 andnucleotide precursors 17 in an aqueous environment. In some embodiments,the reaction chamber 11 is designed to be temperature controlled, forexample by incorporation of Pelletier elements or other methods known inthe art. Methods of controlling temperature for low volume liquids usedin nucleic acid polymerization are known in the art. (See, e.g., U.S.Pat. Nos. 5,038,853, 5,919,622, 6,054,263 and 6,180,372.)

In certain embodiments, the reaction chamber 11 and any associated fluidchannels, for example, to provide connections to a molecule dispenser21, to a waste port, to a template 13 loading port, or to a source ofsynthetic reagent 15 are manufactured in a batch fabrication process, asknown in the fields of computer chip manufacture or microcapillary chipmanufacture. In some embodiments, the reaction chamber 11 and othercomponents of the apparatus 10, such as the molecule dispenser 21, maybe manufactured as a single integrated chip. Such a chip may bemanufactured by methods known in the art, such as by photolithographyand etching. However, the manufacturing method is not limiting and othermethods known in the art may be used, such as laser ablation, injectionmolding, casting, or imprinting techniques. Methods for manufacture ofnanoelectromechanical systems may be used for certain embodiments, suchas those employing a molecule dispenser 21. (See, e.g., Craighead,Science 290:1532-36, 2000.) Microfabricated chips are commerciallyavailable from sources such as Caliper Technologies Inc. (Mountain View,Calif.) and ACLARA BioSciences Inc. (Mountain View, Calif.).

In a non-limiting example, Borofloat glass wafers (Precision Glass &Optics, Santa Ana, Calif.) may be pre-etched for a short period inconcentrated HF (hydrofluoric acid) and cleaned before deposition of anamorphous silicon sacrificial layer in a plasma-enhanced chemical vapordeposition (PECVD) system (PEII-A, Technics West, San Jose, Calif.).Wafers may be primed with hexamethyldisilazane (HMDS), spin-coated withphotoresist (Shipley 1818, Marlborough, Mass.) and soft-baked. A contactmask aligner (Quintel Corp. San Jose, Calif.) may be used to expose thephotoresist layer with one or more mask designs, and the exposedphotoresist removed using a mixture of Microposit developer concentrate(Shipley) and water. Developed wafers may be hard-baked and the exposedamorphous silicon removed using CF₄ (carbon tetrafluoride) plasma in aPECVD reactor. Wafers may be chemically etched with concentrated HF toproduce the reaction chamber 11 and any channels. The remainingphotoresist may be stripped and the amorphous silicon removed.

Access holes may be drilled into the etched wafers with a diamond drillbit (Crystalite, Westerville, Ohio). A finished chip may be prepared bythermally bonding an etched and drilled plate to a flat wafer of thesame size in a programmable vacuum furnace (Centurion VPM, J. M. Ney,Yucaipa, Calif.). In certain embodiments, the chip may be prepared bybonding two etched plates to each other. Alternative exemplary methodsfor fabrication of a reaction chamber 11 chip are disclosed in U.S. Pat.Nos. 5,867,266 and 6,214,246.

To facilitate detection of nucleotide precursors 17 by the detectionunit 12, the material comprising the reaction chamber 11 may be selectedto be transparent to electromagnetic radiation at the excitation andemission frequencies used for the detection unit 12. Glass, silicon, andany other materials that are generally transparent in the frequencyranges used for Raman spectroscopy, fluorescence spectroscopy,luminescence spectroscopy, or other forms of spectroscopy may be usedfor construction of the reaction chamber 11. In some embodiments thesurfaces of the reaction chamber 11 that are opposite the detection unit12 may be coated with silver, gold, platinum, copper, aluminum or othermaterials that are relatively opaque to the detection unit 12. In thatposition, the opaque material is available to enhance the Raman or othersignal, for example by surface enhanced Raman spectroscopy, while notinterfering with the function of the detection unit 12. In alternativeembodiments, a mesh comprising silver, gold, platinum, copper oraluminum may be placed inside the reaction chamber.

In various embodiments, the reaction chamber 11 may have an internalvolume of about 1 picoliter, about 2 picoliters, about 5 picoliters,about 10 picoliters, about 20 picoliters, about 50 picoliters, about 100picoliters, about 250 picoliters, about 500 picoliters, about 1nanoliter, about 2 nanoliters, 5 nanoliters, about 10 nanoliters, about20 nanoliters, about 50 nanoliters, about 100 nanoliters, about 250nanoliters, about 500 nanoliters, about 1 microliter, about 2microliters, about 5 microliters, about 10 microliters, about 20microliters, about 50 microliters, about 100 microliters, about 250microliters, about 500 microliters, or about 1 milliliter.

Molecule Dispenser

The molecular dispenser 21 is designed to release the nucleotideprecursors 17 into the reaction chamber 11. In certain embodiments, themolecule dispenser 21 may release each type of nucleotide precursor 17in equal amounts. In such embodiments, a single molecule dispenser 21may be used to release all four nucleotide precursors 17 into thereaction chamber 11. Other embodiments may require that the rate ofrelease of the four types of nucleotide precursors 17 be independentlycontrolled. In such embodiments, multiple molecule dispensers 21 may beused. In a non-limiting example, four separate molecule dispensers 21may be used, each releasing a single type of nucleotide precursor 17into the reaction chamber 11.

In various embodiments, the molecular dispenser 21 may be in the form ofa pumping device. Pumping devices that may be used include a variety ofmicromachined pumps that are known in the art. For example, pumps havinga bulging diaphragm, powered by a piezoelectric stack and two checkvalves are disclosed in U.S. Pat. Nos. 5,277,556, 5,271,724 and5,171,132. Pumps powered by a thermopneumatic element are disclosed inU.S. Pat. No. 5,126,022. Piezoelectric peristaltic pumps using multiplemembranes in series, or peristaltic pumps powered by an applied voltageare disclosed in U.S. Pat. No. 5,705,018. Published PCT Application No.WO 94/05414 discloses the use of a lamb-wave pump for transportation offluid in micron scale channels. The skilled artisan will realize thatthe molecule dispenser 21 is not limited to the pumps disclosed herein,but may incorporate any design for the measured disbursement of very lowvolume fluids known in the art.

In other embodiments, the molecular dispenser 21 may take the form of anelectrohydrodynamic pump (e.g., Richter et al., Sensors and Actuators29:159-165 1991; U.S. Pat. No. 5,126,022). Typically, such pumps employa series of electrodes disposed across one surface of a channel orreaction/pumping chamber. Application of an electric field across theelectrodes results in electrophoretic movement of charged species in thesample. Indium-tin oxide films may be particularly suited for patterningelectrodes on substrate surfaces, for example a glass or siliconsubstrate. These methods can also be used to draw nucleotide precursors17 into the reaction chamber 11. For example, electrodes may bepatterned on the surface of the molecule dispenser 21 and modified withsuitable functional groups for coupling nucleotide precursors 17 to thesurface of the electrodes. Application of a current between theelectrodes on the surface of the molecule dispenser 21 and an opposingelectrode results in electrophoretic movement of the nucleotideprecursors 17 into the reaction chamber 11.

In certain embodiments, the molecular dispenser 21 may be designed todispense a single nucleotide precursor 17 at a time. In otherembodiments, the molecular dispenser 21 may be designed to dispensenucleotide precursors 17 in volumes of about 1 picoliter, about 2picoliters, about 5 picoliters, about 10 picoliters, about 20picoliters, about 50 picoliters, about 100 picoliters, about 250picoliters, about 500 picoliters, about 1 nanoliter, about 2 nanoliters,5 nanoliters, about 10 nanoliters, about 20 nanoliters, about 50nanoliters, about 100 nanoliters, about 250 nanoliters, about 500nanoliters, about 1 microliter, about 2 microliters, about 5microliters, about 10 microliters, about 20 microliters or about 50microliters

Detection Unit

Embodiments Involving Raman Spectroscopy

In some embodiments, the detection unit 12 is designed to detect andquantify nucleotide precursors 17 by Raman spectroscopy. Various methodsfor detection of nucleotide precursors 17 by Raman spectroscopy areknown in the art. (See, e.g., U.S. Pat. Nos. 5,306,403; 6,002,471;6,174,677). Variations on surface enhanced Raman spectroscopy (SERS) orsurface enhanced resonance Raman spectroscopy (SERRS) have beendisclosed. In SERS and SERRS, the sensitivity of the Raman detection isenhanced by a factor of 10⁶ or more for molecules adsorbed on roughenedmetal surfaces, such as silver, gold, platinum, copper or aluminumsurfaces.

A non-limiting example of a detection unit 12 is disclosed in U.S. Pat.No. 6,002,471. In this embodiment, the excitation beam 20 is generatedby either a Nd:YAG laser 18 at 532 nm wavelength or a Ti:sapphire laser18 at 365 nm wavelength. Pulsed laser beams 20 or continuous laser beams20 may be used. The excitation beam 20 passes through confocal opticsand a microscope objective, and is focused onto the reaction chamber 11.The Raman emission light from the nucleotide precursors 17 is collectedby the microscope objective and the confocal optics and is coupled to amonochromator 19 for spectral dissociation. The confocal optics includesa combination of dichroic filters, barrier filters, confocal pinholes,lenses, and mirrors for reducing the background signal. Standard fullfield optics can be used as well as confocal optics. The Raman emissionsignal is detected by a Raman detector 19. The detector 19 includes anavalanche photodiode interfaced with a computer for counting anddigitization of the signal. In certain embodiments, a mesh comprisingsilver, gold, platinum, copper or aluminum may be included in thereaction chamber 11 to provide an increased signal due to surfaceenhanced Raman or surface enhanced Raman resonance.

Alternative embodiments of detection units 12 are disclosed, forexample, in U.S. Pat. No. 5,306,403, including a Spex Model 1403double-grating spectrophotometer 19 equipped with a gallium-arsenidephotomultiplier tube (RCA Model C31034 or Burle Industries ModelC3103402) operated in the single-photon counting mode. The excitationsource 18 is a 514.5 nm line argon-ion laser from SpectraPhysics, Model166, and a 647.1 nm line of a krypton-ion laser (Innova 70, Coherent).

Alternative excitation sources 18 include a nitrogen laser (LaserScience Inc.) at 337 nm and a helium-cadmium laser (Liconox) at 325 nm(U.S. Pat. No. 6,174,677). The excitation beam 20 may be spectrallypurified with a bandpass filter (Corion) and may be focused on thereaction chamber 11 using a 6×objective lens (Newport, Model L6X). Theobjective lens may be used to both excite the nucleotide precursors 17and to collect the Raman signal, by using a holographic beam splitter(Kaiser Optical Systems, Inc., Model HB 647-26N18) to produce aright-angle geometry for the excitation beam 20 and the emitted Ramansignal. A holographic notch filter (Kaiser Optical Systems, Inc.) may beused to reduce Rayleigh scattered radiation. Alternative Raman detectors19 include an ISA HR-320 spectrograph equipped with a red-enhancedintensified charge-coupled device (RE-ICCD) detection system (PrincetonInstruments). Other types of detectors 19 may be used, such as chargedinjection devices, photodiode arrays or phototransistor arrays.

Any suitable form or configuration of Raman spectroscopy or relatedtechniques known in the art may be used for detection of nucleotides 16including but not limited to normal Raman scattering, resonance Ramanscattering, surface enhanced Raman scattering, surface enhancedresonance Raman scattering, coherent anti-Stokes Raman spectroscopy(CARS), stimulated Raman scattering, inverse Raman spectroscopy,stimulated gain Raman spectroscopy, hyper-Raman scattering, molecularoptical laser examiner (MOLE) or Raman microprobe or Raman microscopy orconfocal Raman microspectrometry, three-dimensional or scanning Raman,Raman saturation spectroscopy, time resolved resonance Raman, Ramandecoupling spectroscopy or UV-Raman microscopy.

Embodiments Involving FRET

In certain alternative embodiments, the nucleotide precursors 17 may beidentified and quantified using fluorescence resonance energy transfer(FRET). FRET is a spectroscopic phenomenon used to detect proximitybetween a donor molecule and an acceptor molecule. The donor andacceptor pairs are chosen such that fluorescent emission from the donoroverlaps the excitation spectrum of the acceptor. When the two moleculesare associated (at a distance of less than 100 Angstroms), theexcited-state energy of the donor is transferred non-radiatively to theacceptor and the donor emission is quenched. If the acceptor molecule isa fluorophore then its emission is enhanced. Compositions and methodsfor use of FRET with oligonucleotides are known in the art (e.g., U.S.Pat. No. 5,866,366).

Molecules that are frequently used as tags for FRET include fluorescein,5-carboxyfluorescein (FAM),2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), rhodamine,6-carboxyrhodamine (R6G), N,N,N′,N′-tetramethyl-6-carboxyrhodamine(TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL), and 5-(2′-aminoethyl)aminonaphthalene-1-sulfonicacid (EDANS). Other potential FRET donor or acceptor molecules are knownin the art (See U.S. Pat. No. 5,866,336, Table 1). The skilled artisanwill be familiar with the selection of pairs of tag molecules for FRET(U.S. Pat. No. 5,866,336).

In embodiments involving FRET, the donor and acceptor molecules may becovalently or non-covalently attached to various constituents of thesequencing apparatus 10. In certain embodiments, the donor or acceptormolecules may be attached to the nucleotide precursors 17, to thetemplate strand 13, or to the polymerase 15.

In certain embodiments, the donor molecule may be attached to thetemplate strand 13 and the acceptor molecules attached to the nucleotideprecursors 17. In this case, each type of nucleotide precursor 17 shouldbe attached to an acceptor molecule with a distinguishable emissionspectrum, while the donor molecule should be selected to have a broademission spectrum that overlaps with the excitation spectra for all fourof the acceptor molecules. Multiple donor molecules will be present onthe template strand 13, for example in the form of fluorescentintercalating agents that insert into double-stranded nucleic acids. Inalternative embodiments, the donor molecules may be covalently attachedto the template strand 13, in a position that does not interfere withbase pair formation. Upon excitation, the multiple donor molecules willtransfer their energy to the acceptor tag molecules attached to thenucleotide precursors 17, resulting in an enhanced emission signal fromthe acceptor molecules. Because the strength of the signal enhancementdecreases rapidly with distance, the greatest signal enhancement willoccur for nucleotide precursors 17 that are incorporated into thenascent strand 16, while nucleotide precursors 17 that are free insolution within the reaction chamber 11 should show relatively weaksignal enhancement. The wavelength of the excitation beam 20 may beselected to maximally excite the donor molecules, while only weaklyexciting the acceptor molecules. In this case, only nucleotideprecursors 17 that are incorporated into the nascent strand 16 willproduce a detectable fluorescent signal. As each nucleotide precursor 17is incorporated into the nascent strand 16, the signal from its donortag will be detected.

In certain embodiments, the template nucleic acid 13 to be sequenced maybe held within the field of view of a fluorescence microscope by methodsknown in the art, for example by use of an optical trap (e.g., U.S. Pat.No. 6,136,543). A non-limiting example of a fluorescence microscope thatmay be used is an inverted phase-contrast and incident-lightfluorescence microscope (IMT2-RFC, Olympus Co., Ltd.), using anoil-immersed 100 power lens (Plan.multidot.Apochromat.times.100, 1.40NA, Olympus Co., Ltd.) The excitation beam 20 may be emitted by a laser18, as discussed above. Fluorescence emission may be collected throughthe objective lens, using appropriate filters, and detected using anysensitive fluorescence detector 19, such as a CCD device, photodiodes,photomultiplier tubes, or the equivalent.

In alternative embodiments, the donor molecule may be attached to thepolymerase 15. As discussed above, each type of nucleotide precursor 17should have a distinguishable acceptor molecule and the emissionspectrum of the donor should overlap the excitation spectra of each ofthe acceptor molecules. Fluorescent detection may be performed asdiscussed in the embodiments involving a donor tagged template nucleicacid 13. Because the number of donor molecules will be substantiallyless than with the template 13 labeling method, the magnitude of signalenhancement for the acceptor molecules should be lower. However, in thisembodiment the fluorescence resonance transfer should be limited tonucleotide precursors 17 that are either at or are close to thecatalytic site of the polymerase 15. The donor molecule should beattached close to the catalytic site, but in a position where it willnot interfere with the polymerase activity of the synthetic reagent 15.In this embodiment, a much less complicated FRET signal should bedetected.

Information Processing and Control System and Data Analysis

In certain embodiments, the sequencing apparatus 10 may comprise aninformation processing and control system. The embodiments are notlimiting for the type of information processing and control system used.An exemplary information processing and control system may incorporate acomputer comprising a bus for communicating information and a processorfor processing information. In one embodiment, the processor is selectedfrom the Pentium® family of processors, including without limitation thePentium® II family, the Pentium® III family and the Pentium® 4 family ofprocessors available from Intel Corp. (Santa Clara, Calif.). Inalternative embodiments, the processor may be a Celeron®, an Itanium®,or a Pentium Xeon® processor (Intel Corp., Santa Clara, Calif.). Invarious other embodiments, the processor may be based on Intel®architecture, such as Intel® IA-32 or Intel® IA-64 architecture.Alternatively, other processors may be used.

The computer may further comprise a random access memory (RAM) or otherdynamic storage device, a read only memory (ROM) and/or other staticstorage and a data storage device such as a magnetic disk or opticaldisc and its corresponding drive. The information processing and controlsystem may also comprise other peripheral devices known in the art, sucha display device (e.g., cathode ray tube or Liquid Crystal Display), analphanumeric input device (e.g., keyboard), a cursor control device(e.g., mouse, trackball, or cursor direction keys) and a communicationdevice (e.g., modem, network interface card, or interface device usedfor coupling to Ethernet, token ring, or other types of networks).

In particular embodiments, the detection unit 12 may also be coupled tothe bus. Data from the detection unit 12 may be processed by theprocessor and the data stored in the main memory. Data on emissionprofiles for standard nucleotide precursors 17 may also be stored inmain memory or in ROM. The processor may compare the emission spectrafrom nucleotide precursors 17 in the reaction chamber 11 to identify thetype of nucleotide precursor 17 incorporated into the nascent strand 16.The main memory may also store the sequence of nucleotide precursors 17disappearing from the reaction chamber 11. The processor may analyze thedata from the detection unit 12 to determine the sequence of thetemplate nucleic acid 13.

It is appreciated that a differently equipped information processing andcontrol system than the example described above may be used for certainimplementations. Therefore, the configuration of the system may vary indifferent embodiments. It should also be noted that, while the processesdescribed herein may be performed under the control of a programmedprocessor, in alternative embodiments, the processes may be fully orpartially implemented by any programmable or hardcoded logic, such asField Programmable Gate Arrays (FPGAs), TTL logic, or ApplicationSpecific Integrated Circuits (ASICs), for example. Additionally, themethod may be performed by any combination of programmed general purposecomputer components and/or custom hardware components.

Following the data gathering operation, the data will typically bereported to a data analysis operation. To facilitate the analysisoperation, the data obtained by the detection unit 12 will typically beanalyzed using a digital computer. Typically, the computer will beappropriately programmed for receipt and storage of the data from thedetection unit 12, as well as for analysis and reporting of the datagathered. In certain embodiments, this may involve determining theconcentration of nucleotide precursors 17 in the reaction chamber 11from the Raman data and subtracting background Raman signals.

In certain embodiments, the information processing and control systemmay control the amount of nucleotide precursors 17 that are dispensedinto the reaction chamber 11. In such embodiments, the informationprocessing and control system may interface between the detection unit12 and the molecule dispenser 21, to regulate the release of nucleotideprecursors 17 by the molecule dispenser 21 to approximately match therate of incorporation of nucleotide precursors 17 into the nascentstrand 16.

In certain embodiments, custom designed software packages may be used toanalyze the data obtained from the detection unit 12. In alternativeembodiments, data analysis may be performed, using an informationprocessing and control system and publicly available software packages.Non-limiting examples of available software for DNA sequence analysisinclude the PRISM™ DNA Sequencing Analysis Software (Applied Biosystems,Foster City, Calif.), the Sequencher™ package (Gene Codes, Ann Arbor,Mich.), and a variety of software packages available through theNational Biotechnology Information Facility at websitewww.nbif.org/links/1.4.1.php.

1. A method of sequencing nucleic acid molecules comprising: a)inserting multiple copies of a template nucleic acid molecule into areaction chamber; b) synthesizing a complementary nucleic acid moleculefrom nucleotide precursors with a polymerase; c) monitoring the order ofincorporation of nucleotide precursors into the complementary nucleicacid molecule by surface enhanced Raman scattering, surface enhancedresonance Raman scattering, stimulated Raman scattering, inverse Raman,stimulated gain Raman spectroscopy, hyper-Raman scattering or coherentanti-Stokes Raman scattering, wherein a tag molecule is attached to eachnucleotide precursor; and d) determining the sequence of the templatenucleic acid molecule from the order of incorporation of nucleotideprecursors into complementary nucleic acid molecules.
 2. The method ofclaim 1, wherein the polymerase is a DNA polymerase.
 3. The method ofclaim 2, further comprising adding a primer, wherein the primer iscomplementary in sequence to a portion of the template nucleic acidmolecule.
 4. The method of claim 3, wherein the primer is complementaryto the 3′ end of the template nucleic acid molecule.
 5. The method ofclaim 1, wherein the template nucleic acid molecules are attached to animmobilization surface.
 6. The method of claim 5, wherein the templatenucleic acid molecules are attached to the immobilization surfacethrough a linker arm.
 7. The method of claim 3, wherein the primer isattached to an immobilization surface.
 8. The method of claim 1, whereineach type of nucleotide precursor is attached to a distinguishable tagmolecule.
 9. The method of claim 8, wherein tag molecules are selectedfrom the group consisting of TRIT (tetramethyl rhodamine isothiol), NBD(7-nitrobenz-2-oxa-1,3-diazole), Texas Red dye, phthalic acid,terephthalic acid, isophthalic acid, cresyl fast violet cresyl blueviolet, brilliant cresyl blue, para-aminobensoic acid, erythroisine,aminoacridine, cyanide, thiol, chlorine, bromine, methyl, phosphorus,sulfur and carbon nanotubes.
 10. The method of claim 1, wherein tagmolecules are selected from the group consisting of TRIT (tetramethylrhodamine isothiol), NBD (7-nitrobenz-2-oxa-1,3-diazole), Texas Red dye,phthalic acid, terephthalic acid, isophthalic acid, cresyl fast violetcresyl blue violet, brilliant cresyl blue, para-aminobensoic acid,erythroisine, aminoacridine, cyanide, thiol, chlorine, bromine, methyl,phosphorus, sulfur and carbon nanotubes.
 11. The method of claim 1,wherein the polymerase is a DNA polymerase, an RNA polymerase or areverse transcriptase.
 12. The method of claim 1, wherein a surface ofthe reaction chamber is coated with silver, gold, platinum, copper, oraluminum.
 13. The method of claim 12, wherein the coated surface of thereaction chamber is opposite a detection unit.